Project Summary/Abstract Every year, ~900,000 Americans are diagnosed with complications from clot formation leading to ~300,000 deaths and ~$28 billion annual cost. The number of adults with clot-related complications are estimated to reach ~1.82 million by 2050. Blood clots form through the coagulation cascade and are part of the natural response to injury and cut, preventing loss of blood from severed blood vessels. However, the life-saving process of clot formation can become life-threatening if a blood vessel is blocked due to a clot. Indeed, clot-related pathologies including heart attack, stroke, and pulmonary embolism account for ~50% of all hospital deaths. Reperfusion, the act of restoring blood flow, with tissue plasminogen activator (tPA) is the gold standard treatment for ischemic stroke. Adverse side effects and cost of tPA motivate alternative approaches to treat clots. A major component of blood clots is crosslinked fibrin. Water-soluble fibrin monomers assemble into water-insoluble fibrin polymer, which makes them difficult to study using common biochemical assays. Human matrix metalloproteases (MMPs) present in blood have fibrinolytic activity and are therefore potentially alternative targets in blood clot management. MMPs are a family of 25 enzymes and are implicated in the majority of the top ten causes of death. MMP1 is the best studied member of the MMP family with a crystal structure available and details of its interaction with collagen known. We have strong evidence for fibrinolytic activity of MMP1, which is modulated by antibiotics and MMP9, another important MMP-family member that is upregulated in many disease. We propose to define the mechanism of fibrin degradation at the single molecule with synergistic applications of ensemble assays and molecular dynamics simulations.